Comparative in vitro digestibility of human milk and infant formulas: In-depth simulation of macronutrient breakdown and gut microbiota response

Breastfeeding practices are often impeded by multifactorial circumstances, leading to the need for infant formulas. Previous studies have determined the differences in performance between human milk (HM) and infant formulas (IFs) in terms of macronutrient digestibility, impact on colonic microbiota, and immunomodulatory effects. However, few have adopted a comprehensive approach to investigate above-reported issues, integrating digestion, lipidomics, proteomics, immunomodulation, colonic fermentation and microbiota outcomes in infants. In vitro gastrointestinal digestion was carried out following the INFOGEST-adapted breastfeed infant-specific protocol, followed by static in vitro colonic fermentation. Lipolysis and proteolysis were evaluated, alongside changes in particle size distribution, molecular characterization of proteins, lipids and polar compounds, and microbial targeted profile (qPCR). Additionally, the immunomodulatory effect was studied using a TLR4 reporter cell line. Milk lipids were hydrolysed differently during gastric and intestinal stages, but total lipolysis was similar in both infant formulas and human milk. The infant formula subjected to 1-year storage was characterized by higher concentration of modified proteins and peptides and excelled during colonic fermentation due to an induced increase in Bifidobacterium and Bacteroides. Furthermore, independently of the storage effects, both infant formulas did not induce TLR4 activation, unlike the control cow milk and HM. Although IFs and HM differed in digestive kinetics and certain bioactive responses, both exhibited convergent trends in overall digestion and fermentation profiles, suggesting partial functional similarity but not biological equivalence.